Motor innervation has a trophic influence on skeletal muscle fibers. If the nerve supply to a muscle is compromised, as occurs in certain muscular dystrophies or after surgical denervation, the muscle atrophies. Muscle wasting is also a consequence of insulin deficiency, and can be corrected only b replacing the hormone. When a skeletal muscle is denervated, it rapidly loses its ability to respond to insulin. This loss may be an important factor in the muscle wasting that occurs after denervation. Skeletal muscle cells in culture are also resistant to insulin, possibly because they are not innervated. A long-term objective of this project is to define the mechanisms involved in the insulin-resistance which develops when a skeletal muscle is denervated in vivo and which is found in uninnervated skeletal muscle cell in vitro. There is evidence that a decrease in the activity of Type I protein phosphatase, due to increased activity of phosphatase inhibitor-1, occurs after denervation. One hypothesis is that the inability of insulin to stimulate protein dephosphorylation in denervated muscle and cultured muscle cells is due to a deficiency in Type I protein phosphatase. The following specific aims address this hypothesis, would provide useful information concerning the role of cAMP-dependent protein kinase (cAdPK) and Type I protein phosphatase in skeletal muscle cells even if the hypothesis proves to be incorrect. Aim 1: Attempt to overcome the phosphatase deficiency in cultured muscle cells by expressing Type I phosphatase. We will select cell lines after transfecting L6 myoblasts with DNA encoding the enzyme. Aim 2: Investigate the effect of phosphatase expression by measuring changes in the phosphorylation states of substrates for Type I protein phosphatase, and by assessing insulin actions on activating glycogen synthase and increasing glucose incorporation into glycogen. Aim 3: Prepare muscle cells lines deficient in cAdPK by transfecting cells with cDNA encoding a mutant R subunit of cAdPK. This kinase is necessary for inhibitor-1 activity, and decreasing its activity would be expected to increase Type I phosphatase activity. Aim 4: Analyze the effects of down-regulating cAdPK on muscle cell function by investigating the effects of kinase depletion on hormonal responsiveness and phosphorylation states of interconvertible enzymes. Aim 5: Investigate the effect of depleting cAdPK and increasing Type I phosphatase in the same cells by using hybrid myotubes. Aim 6: Generate transgenic mice expressing mutant R subunits to try to down-regulate cAdPK in vivo. Aim 7: Analyze skeletal muscles from transgenic animals. Activation of phosphorylase and inactivation of glycogen synthase in response to epinephrine are pathways involving cAdPK. Functional deficiency of cAdPK in muscles of the transgenic animals will be documented by the absence of these responses to epinephrine. Down-regulation of the kinase would be expected to decrease phosphorylation of phosphatase inhibitor-1, and might prevent the loss of insulin activation of glycogen synthase that occurs after denervation.